Gene duplication in the major insecticide target site,Rdl, in Drosophila melanogasterEmily J. Remnanta,b,1, Robert T. Gooda, Joshua M. Schmidta, Christopher Lumba, Charles Robina, Phillip J. Daborna,and Philip Batterhama

aDepartment of Genetics and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC 3010, Australia; and bBehaviourand Genetics of Social Insects Laboratory, School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia

Edited by Trudy F. C. Mackay, North Carolina State University, Raleigh, NC, and approved July 23, 2013 (received for review June 13, 2013)

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloridechannel subunit that is targeted by cyclodiene and phenylpyrazoleinsecticides. The gene was first characterized in Drosophila mela-nogaster by genetic mapping of resistance to the cyclodiene diel-drin. The 4,000-fold resistance observed was due to a single aminoacid replacement, Ala301 to Ser. The equivalent change was subse-quently identified in Rdl orthologs of a large range of resistant in-sect species. Here, we report identification of a duplication at the Rdllocus in D. melanogaster. The 113-kb duplication contains one WTcopy of Rdl and a second copy with two point mutations: an Ala301

to Ser resistance mutation and Met360 to Ile replacement. Individualswith this duplication exhibit intermediate dieldrin resistance com-pared with single copy Ser301 homozygotes, reduced temperaturesensitivity, and altered RNA editing associated with the resistantallele. Ectopic recombination between Roo transposable elementsis involved in generating this genomic rearrangement. The duplica-tion phenotypes were confirmed by construction of a transgenic,artificial duplication integrating the 55.7-kb Rdl locus with a Ser301

change into an Ala301 background. Gene duplications can contributesignificantly to the evolution of insecticide resistance, most com-monly by increasing the amount of gene product produced.Here however, duplication of the Rdl target site creates perma-nent heterozygosity, providing unique potential for adaptivemutations to accrue in one copy, without abolishing the endoge-nous role of an essential gene.

The single point mutation in the Resistance to dieldrin (Rdl)gene represents one of the most significant cases of target site

resistance to an insecticide yet observed. Cyclodiene resistancewas reported in 62% of insecticide resistant species in the 1980s,following widespread use of cyclodiene insecticides, includingdieldrin, which started in the 1950s (1). The nature of the genetictarget, Rdl, was discovered after dieldrin was discontinued be-cause of the widespread evolution of resistance in many species.Rdl was first discovered in Drosophila melanogaster using a posi-tional cloning approach. High homology to human GABA recep-tors confirmed it was the first insect ligand-gated chloride channelsubunit identified (2–4). A point mutation in the chloride channelpore-lining domain, replacing alanine 301 with serine, was presentin all resistant D. melanogaster strains (5). This mutation provided4,000-fold resistance when homozygous and lower levels of re-sistance in heterozygotes (2, 6). The homologous mutation wassubsequently found in a large number of cyclodiene-resistant spe-cies from many insect orders (7–9), as well as a glycine replacementat the homologous site in some resistant strains of Drosophilasimulans and other species (5, 10, 11).Characterization of deficiency lines and inversions in D. mela-

nogaster showed that Rdl is an essential gene (3). Thus, the Ala301 toSer or Gly mutation in Rdl exhibits unique properties, providinghigh levels of dieldrin resistance without abolishing the role ofthe RDL receptor (12). Electrophysiological studies showed thatthe 301 replacement affects cyclodiene sensitivity by two mech-anisms: inhibiting direct binding and allosterically modifyingthe Rdl receptor to disrupt the antagonist-favored conformation(13). These differences in channel properties have little effecton overall fitness in D. melanogaster other than temperature

sensitivity. Resistant adults exposed to temperature stress (38 °C)showed delayed recovery indicated by temporarily impairedflight (14). Laboratory studies of D. melanogaster and D. simulansshowed no decline in Ser or Gly301 allele frequencies in pop-ulation cages after 1 y in the absence of cyclodiene insecticideselection (15). More recently, the Ala301 to Ser change wasshown to decrease sleep latency (16). Because fitness testing wasconducted in the laboratory, the true extent of fitness costs mayhave been underestimated. Conversely, in the blowfly Luciliacuprina, field studies have shown that in the absence of dieldrin,the Rdl resistance allele is at a dramatic selective disadvantage(17). Resistant individuals were more severely selected againstduring overwintering than other points throughout the year (18).RDL is highly conserved in insects, and the universal nature of

the Ala to Ser/Gly resistance mutation exemplifies this conser-vation. However, an influx of genomic information from insectspecies has shown that some lineages have multiple Rdl loci, withthree copies present in Lepidopteran genomes and two presentin the aphid Acyrthosiphon pisum (19–21). Before sequencing theA. pisum genome, two Rdl copies were reported in the peachpotato aphids Myzus persicae and M. nicotinae (10). The presenceof two copies in all three species suggests it is an ancient duplicationpresent in the ancestral aphid lineage.Gene amplification has previously been implicated as a major

evolutionary avenue to attaining insecticide resistance, primarily inthe form of increased gene expression of detoxification enzymes(22). Organophosphorus chemical resistance in the mosquito,Culex pipiens, is mediated by esterase overproduction due toregulatory changes and 250-fold increased copy number of ester-ase B3 (23). In D. melanogaster, an allelic series at the cytochromeP450 Cyp6g1 locus involves a gene duplication and a variety oftransposable element (TE) insertions. The most derived alleles arecorrelated with increased enzyme production and multiinsecticideresistance, including dichlorodiphenyltrichloroethane (DDT) (24).Here we report the occurrence of a recent duplication span-

ning the genomic region of Rdl in D. melanogaster. Because Rdl isan essential gene (3) and a major insecticide target site forcyclodiene and phenylpyrazole insecticides, copy number varia-tion allows for evolutionary flexibility, where adaptive mutationsmay accumulate in one copy, whereas WT function is maintainedin the other. The duplication creates a situation of permanentheterozygosity, providing advantages in the presence and absenceof insecticide.

ResultsIdentification of the Rdl Gene Duplication. We examined the avail-able genome sequence for the Rdl gene in 168 D. melanogasterlines [The Drosophila Genomic Reference Panel (DGRP) (25)].

Author contributions: E.J.R., P.J.D., and P.B. designed research; E.J.R., R.T.G., C.L., andP.J.D. performed research; E.J.R., R.T.G., J.M.S., and C.L. contributed new reagents/analytictools; E.J.R., R.T.G., J.M.S., and C.R. analyzed data; and E.J.R., C.R., and P.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: emily.remnant@sydney.edu.au.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311341110/-/DCSupplemental.

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The DGRP was established in 2003 from an outbred populationcollected at a farmers market in Raleigh, NC. Isofemale lineswere inbred for 20 generations (25). Among the inbred lines,there were 10 nonsynonymous polymorphisms in Rdl (Table S1;Fig. S1). The Rdl resistance mutation, Ser301, was present in fourlines (Ral-317, Ral-318, Ral-378, and Ral-491); however, in thefirst three, the mutation was heterozygous with Ala. These threelines also contained a second heterozygous, nonsynonymous mu-tation, Met360 to Ile. Met360 is one of four RNA-editing locationsin Rdl. Editing to Val occurs in 10% of adult head RdlBD tran-scripts, where RdlBD is the most common splice isoform of fouralternately spliced transcripts (26).Genomic heterozygosity within an inbred line may indicate copy

number variation (27). We confirmed Ala301/Ser301 heterozygosityby HaeII restriction digest of PCR products from Ral-318 andRal-378 individuals (Materials and Methods; Fig. S2). Residualheterozygosity still occurs in these inbred lines (25), so to dis-tinguish this from our prediction that the Rdl gene was dupli-cated, we followed the inheritance of the variants. If they werealleles, alternate segregation would be observed, whereas if itwas a duplication, they would cosegregate. Ral-318 and Ral-378were crossed to an Rdl WT line, w1118. F1 offspring were testedfor genotype at the 301 site using the HaeII restriction di-gestion assay. One hundred percent were heterozygous for theAla301/Ser301 polymorphism (n = 32; Fig. S2), indicating cose-gregation of alleles and supporting the duplication hypothesis.The assembled genome sequence Ral-318 and Ral-378 showed

regions of heterozygosity spanning ∼110 kb surrounding Rdl.Genome coverage showed a greater number of sequence readsacross the putative duplication region compared with adjacentregions of the 3L chromosome arm (Fig. 1). Alignment of reads inthe region revealed the putative duplication arrangement. The firstbreakpoint occurred at the site of a Roo TE long terminal repeat(LTR) in intron 7 of nervous wreck (nwk), the gene directly 5′ ofRdl. The second breakpoint occurred 113 kb downstream, thesite of a second Roo TE LTR in intron 1 of the glutamate receptorIB (Glu-RIB) locus. The rearrangement results in two tandemlyarranged copies of Rdl and surrounding genes within the 113-kbduplication (Fig. 2). Five genes were fully duplicated across the1-kb region, as well as partial duplication of the 3′ portion of nwkand the 5′ portion of Glu-RIB (Fig. 2). Including Rdl Ala301/Serand Met360/Ile, 15 heterozygous nonsynonymous differences wereidentified between the duplicated regions (Table S2).Six primers were designed to regions of nwk, Glu-RIB, and the

Roo LTR (Table S3). Combinations therein and sequencing ofpositive products confirmed the predicted duplication topology.The presence of an internal Roo element within the duplicationstructure was confirmed with long PCR spanning the Glu-RIBintron 1 to nwk intron 7 (Fig. S3; Fig. 2). A feature of Roo TEs isinsertion site duplication, where 5 bp of genomic DNA is du-plicated at either side of the point where the TE inserts (28).When ectopic recombination occurs between two different TEinsertions, the flanking sequences should differ on each side ofthe TE. We examined the genomic sequence in Ral-317, -318,and -378 at both Roo insertion sites. We found the nwk-Roo hada different 5-bp insertion site duplication to the Glu-RIB-Roo(AATCT and ACCTG, respectively).The primers designed to detect the nwk-Roo TE also amplified

a product from the RdlR MD-RR line, first isolated in 1990(29), suggesting this line carried a Roo TE LTR at this site.Sequencing this product showed the same 5-bp target site du-plication adjacent to the Roo LTR (AATCT) as Ral-317, -318,and -378. Previous characterization of RdlR MD-RR showed thatit contained both Ser301 and Ile360 replacements (6), indicatinga common haplotype to the Ral lines. In contrast, the nwk-Rooproduct was not amplified in another single-copy Ser301 line fromthe DGRP, Ral-491, nor did this line contain the Met360 to Ilereplacement. Long PCR was performed on RdlR-MDRR to assesswhether a duplication was also present, although we were unableto amplify the nwk-Roo-GluRIB product (Fig. S3). Sequencingof introns and cDNA from multiple regions of Rdl revealed no

heterozygous SNPs, confirming it was homozygous for Ser301 andlacked extensive heterozygosity as seen in the duplication lines.

Both Copies of Rdl Are Expressed. cDNA was produced from RNAextracted from heads of 1-d-old adults from Ral-318 and -378.The Rdl exon 7 and 8 region, spanning the 301 and 360 sites,was amplified and cloned. Forty-five individual clones were se-quenced to identify whether both copies of Rdl were expressedand if the two derived nonsynonomous variants were found in thesame copy. This tissue and life stage were chosen to determinewhether Met360 to Val RNA editing still occurred correctly inIle360 mutants (26).The clones fell into two categories: one characterized by the

presence of the Ser301 and Ile360 mutations in the same transcript,and the other category was WT, indicating that both copies ofduplicated Rdl are expressed. The frequency of sequences con-taining the Ser301Ile360 mutations was 24 of 45 (53%), indicatingapproximately the same expression level as the WT copy (21 of45, 47%; χ2 = 0.2; P = 0.655; Table S4). The expressed clonesshowed variation in RNA editing between the two copies (Fig. 3;Table S5). Editing of codon 360 to Val occurred in Ser301Ile360

mutants in 6 of 24 mutant clones as opposed to 1 of 21 WTclones, although this difference was not significant (Fisher exacttest, P = 0.10). There was a significant loss of Asn294 to Aspediting in the Ser301Ile360 mutant, with 12 clones edited at the294 codon in WT, but only 1 edited in mutant clones (Fisher exacttest, P = 0.00013).

Resistance to Dieldrin. Dieldrin toxicity assays were conducted onadults from three naturally derived lines: RdlR MD-RR (singlecopy; Ser301); Ral-378 (duplication; Ala301Met360/Ser301Ile360); andRal-882, (single copy; WT Rdl). Dosage–mortality curves were

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Fig. 1. Genome sequence coverage of 1 Mb of Chr3L spanning the Rdllocus in DGRP sequenced lines. (A) Duplication line Ral-318 and (B) singlecopy line Ral-882. Values are normalized against the average read depthfor each line. The black arrow indicates the approximate position of Rdl.Fold coverage is greater than 1 across the putative duplication region forRal-318 compared with the single copy line 882. Similar results were observedfor Ral-378.

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generated (Fig. 4A). Compared with WT, RdlR showed >45,000-fold resistance to dieldrin. Ral-378, containing both Rdl geno-types, showed significant resistance, with 27-fold survival overWT (Table S6).To isolate the contribution of the Rdl Ser301 mutation to re-

sistance from within the 113-kb genomic duplication, a trans-genic model was generated using the P[acman]-attB vector. Thetransgenic construct incorporated 55.7 kb of Rdl genomic DNA,but excluded all other genes present in the duplication. Both WTAla301 and mutant Ser301 lines were generated in a controlledgenetic background, differing by only a single base pair mutation.Three lines were tested for dieldrin resistance: Φ-86Fb-empty[attP 86Fb line (30)]; Φ-86Fb-P[ac]-RdlWT (Rdl Ala301 insertion);and Φ-86Fb-P[ac]-RdlA301S (Rdl Ser301 insertion). Thus, the insertlines replicated the Rdl duplication, containing two copies of Rdl:one transgenic and one endogenous copy.Φ-86Fb-empty and Φ-86Fb-P[ac]-RdlWT lines showed no differ-

ence in survival (Fig. 4B; Table S6), indicating an extra copy of WTRdl did not modify resistance in the transgenic Φ-86Fb-P[ac]-RdlWT

line. However, Φ-86Fb-P[ac]-RdlA301S had increased survival, withsixfold resistance to dieldrin (Fig. 4B; Table S6). The moderateresistance observed highlighted the semidominant nature of Rdlresistance (2), with combined effects of transgenic Ser301 andendogenous WT Ala301.

Recovery from Heat Shock. To examine temperature sensitivity as-sociated with the Rdl Ser301 mutation (14), heat shock recoverytests were conducted in lines derived from natural populations andtransgenic lines examined previously for dieldrin resistance. Adultfemales were exposed to 38 °C for 10 min, and recovery time wasobserved. Among the naturally derived lines, genotype had a sig-

nificant affect on recovery time [F(2,6) = 41.861, P < 0.0001].Pairwise comparison showed a significant difference betweenWT Ral-882 and Ral-378 (Ala301Met360/Ser301Ile360) (P = 0.004)and Ral-882 and RdlR MD-RR (Ser301) (P < 0.0001), with the Ral-378 and RdlR comparison yielding P = 0.051 (Bonferroni cor-rection). RdlR was the slowest to recover from heat shock, with amedian recovery time of 10 min. Ral-378 showed an interme-diate recovery of 7 min, and WT Ral-882 showed the fastestrecovery time (4 min; Fig. 5A). Genotype also significantly affectedrecovery time in the transgenic lines [F(2,6) = 19.767, P = 0.002].Φ-86Fb-empty and Φ-86Fb-P[ac]-RdlWT were not significantlydifferent from each other (P = 0.585); however, Φ-86Fb-P[ac]-RdlA301S had a significantly increased temperature recovery timecompared with both controls (P = 0.011 and P = 0.003, respectively;Fig. 5B), with a median recovery time of 5.5 min compared with4.5 (Φ-86Fb-empty) and 3.5 min (Φ-86Fb-P[ac]-RdlWT).

DiscussionInvestigating the genome sequence of 168 naturally derived in-bred lines of D. melanogaster revealed a 113-kb tandem dupli-cation encompassing the major insecticide target site, Rdl. Twononsynonymous polymorphisms were present between the du-plicated copies: at the insecticide resistance site, Ala301 to Ser,and an RNA-edited site, Met360 to Ile. Both mutations were foundin the same copy, expressed at equivalent levels to the WT copy.The duplication and associated polymorphisms have implicationsin posttranscriptional RNA editing, dieldrin resistance, and heatshock recovery.When resistance is dominant, heterozygotes often present

intermediate phenotypes in both fitness and resistance to in-secticide. This offset may allow resistant alleles to persist inpopulations in the absence of insecticide (31). Duplications en-able the maintenance of permanent heterozygosity and havepreviously been shown to modify fitness in insecticide resistancecontext. In Culex pipiens, a point mutation in the acetylcholineesterase-1 (Ace-1) locus is associated with high levels of resistanceto organophosphates; however, this mutation reduces Ace-1 ac-tivity by 60%, incurring a significant fitness cost (22). Combininga resistant and susceptible allele by gene duplication offsets partof the fitness cost (32). A fitness offset may also be the case forRdl in the duplicated lines, which show expression of both theresistant Ser301Ile360 copy and the Ala301Met360 WT copy atequal levels and subsequently display intermediate levels of re-sistance (Fig. 4) and heat shock recovery (Fig. 5). Given that D.melanogaster is not generally considered to be a pest, it is likelyto be incidentally exposed to insecticides at a lower frequencyand concentrations that would be the case for pest species. Theintermediate resistance associated with the duplication may beprotective against such exposure.

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Fig. 2. Topology of the duplication. (A) Genomic structure surrounding the Rdl locus in reference single-copy strains. (B) Duplication structure in Ral-317, -318,and -378. The 5′ duplication breakpoint occurs within intron 7 of nwk, the gene directly upstream of Rdl; 113 kb of sequence is duplicated, ending in intron 1of Glu-RIB. Five genes are completely duplicated (Table S2), and the 3′ portion of nwk and 5′ portion of Glu-RIB are partially duplicated. Between the partialGlu-RIB and nwk segments remains a Roo TE, as indicated by LTRs and long PCR products (Fig. S3). The region surrounding the Roo TE is shown in greaterdetail, indicating position of primer binding sites (Table S3).

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Fig. 3. RNA editing frequency. Frequency of RNA editing at three of thefour known editing sites (Ile283 to Val, Asn294 to Asp, Met360 to Val) in Rdltranscripts within the amplified exon 7–8 region, spanning codons 301 and360. (A) Editing in WT Ala301Met360 sequenced clones compared with (B)mutant Ser301Ile360 clones. Frequencies are shown in Tables S4 and S5.

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Gene duplications associated with insecticide resistance havebeen predominantly involved in increased gene expression ofmetabolic enzymes resulting in enhanced insecticide detoxi-fication (24, 33, 34). However, beyond enhanced expression,gene duplication in a target site provides a unique opportunityfor the redundant copy of the duplicate pair to accumulatemutations. Some mutations may be adaptive in certain environ-mental scenarios that are otherwise detrimental to the originalfunction of the gene (33). Gene duplications are one of themajor adaptive forces in eukaryote evolution, but the persistenceof duplicate copies is not favored unless each copy acquires aspecific role. After duplication occurs, the majority of duplicatepairs result in one gene undergoing rapid deleterious mutationsleading to pseudogenization (35). However, a duplicate pair ispreserved if beneficial mutations accrue. Either one memberacquires mutations that provide a new function (neofunction-alization) or the two copies share the original function of theprogenitor gene by subfunctionalizing (35, 36).Duplications may arise via a number of mechanisms including

unequal crossing over or replication slippage (32). For the Rdlduplicated lines, our data suggest ectopic recombination inneighboring Roo TEs initiated the genome rearrangement. TEsand genome rearrangements have previously been implicated inadaptation to environmental pressures such as insecticide resistance(24, 37, 38). TEs contribute substantially to adaptive evolution andhave the capacity to generate deletions, duplications, and regulatorychanges with wide-ranging phenotypic effects that cannot be ach-ieved by point mutations (39). Roo elements are the most commonTE in the D. melanogaster genome (40), and are frequent initiatorsof chromosomal rearrangements such as duplications and deletions(41). PCR analysis indicated a Roo LTR was present in intron 7of nwk in the original RdlR MD-RR line. The RdlR haplotype maytherefore be implicated as a precursor for the duplication.Recombination between the RdlR nwk-Roo haplotype anda downstream Glu-RIB-Roo haplotype would result in thegeneration of the 113-kb tandem arrangement we observe in Ral-317, -318, and -378 (Fig. 6). Observation of different insert siteduplication sequences flanking the Roo LTRs supports our theoryof ectopic recombination between two different Roo TEs ingenerating this duplication structure. Our diagnostic PCRs detectupstream nwk-Roo elements at a low frequency in populations ofD. melanogaster from Australian populations, although nononduplicated populations have yet been identified with thecorresponding downstream Glu-RIB-Roo (SI Text).

Permanent Heterozygosity, Resistance, and Temperature Sensitivity.A functional GABA receptor consists of five subunits. Pentamersformed in homozygous Ser301 mutants contain only resistantsubunits; however, in duplication lines, Ala301 and Ser301 allelesfrom the two Rdl copies would result in heteromeric RDL re-ceptors containing a mixture of resistant and susceptible subunits.The same receptor composition also occurs in Φ-86Fb-P[ac]-RdlA301S, containing transgenic Ser301 and endogenous Ala301Rdl, and is functionally equivalent to single copy Ala301/Serheterozygotes, with heteromeric receptors and semidominant,intermediate resistance levels (2).The role of Ser301 in resistance is well established (5, 6). How-

ever, to distinguish it as the causal mutation for the phenotypesassayed, transgenic lines containing a 55.7-kb Rdl-only duplica-tion were generated. The transgenic construct eliminated othergenes and mutations contained in the 113-kb natural duplication,and isolated the Ser301 mutation in the absence of other non-synonymous replacements including Ile360, present in duplicationlines and the original RdlR MD-RR line (6). A distinct resistanceand temperature sensitive phenotype emerged, even when elimi-nating background factors in the natural populations, which mayconsist of many generations of coadapted modifications. The14 other nonsynonymous changes in the 113-kb duplication, orindeed other genes elsewhere in the genome, may have an additiveeffect on the two phenotypes assayed here in Ral-378. Addition-ally, reduced RNA editing efficiency at codon 294, and increasedediting at codon 360 (Fig. 3; Table S5) may affect RDL receptorproperties and phenotypes alongside the Ser301 and Ile360 muta-tions. However, the contribution of Ser301 is verified by thetransgenic experiments, where the Rdl insertion lines differ by asingle base pair and illustrate the significance of this point mu-tation in genetically identical lines.

Rdl Copy Number Variation in Insects. Previous studies reported twocopies of Rdl in the peach-potato aphid,Myzus persicae: one copywith serine at the equivalent 301 site and the other containingalanine. Cyclodiene resistance was attributed to a glycine changein the alanine copy (10). The recent release of the pea aphid(A. pisum) genome also revealed two copies of Rdl, with Ala inone copy and Ser in the other (21). In Lepidoptera, three copiesof Rdl are present, and in Bombyx mori these copies share 75–91%

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Fig. 4. Dosage mortality response curves for dieldrin. (A) Natural pop-ulations: Ral- 882 (WT, Ala301Met360); Ral-378 (duplication, Ala301Met360/Ser301Ile360); and RdlR MD-RR (single copy, Ser301Ile360). (B) Transgenic lines:Φ-86Fb-empty control, Φ-86Fb-P[ac]-RdlWT (WT Rdl Ala301 insertion) andΦ-86Fb-P[ac]-RdlA301S (mutant Rdl Ser301 insertion). Fold changes are shownin Table S5.

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Fig. 5. Recovery from heat shock. (A) Ral-882 (WT); Ral-378 (duplication,Ala301Met360/Ser301Ile360); and RdlR MD-RR (single copy, Ser301). Three in-dividual replicates are graphed with the average (±SEM) superimposed foreach line. (B) Transgenic lines: Φ-86Fb-empty, Φ-86Fb-P[ac]-RdlWT, andΦ-86Fb-P[ac]-RdlA301S.

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similarity at the protein level (19). However, one copy has Ala,one has Ser, and the third has a glutamine at the equivalent 301site (Fig. S4). This site is therefore polymorphic in species withmultiple copies of Rdl, suggesting a reduction in functionalconstraint within an otherwise highly conserved domain. It mayexplain to some extent why the 301 site is amenable to resistancemutations in single copy species.In Drosophila, this is the first example of copy number variation

at the Rdl locus. Identification of the present D. melanogasterduplication was facilitated by the availability of high-depth ge-nome sequences of a large number of inbred lines expected tobe homozygous. We did not detect the duplication in a survey ofAustralian populations (SI Text) or in the genome sequencesavailable from 139 African D. melanogaster lines (27), suggestinga rare event led to the arrangement discovered in the DGRP. Itis possible that other duplications exist at the Rdl locus, whichwere not detectable by our specific diagnostic PCRs.Since the cessation of cyclodiene insecticide use, the frequency

of the Rdl Ser301 resistance allele varies from undetectable levelsin houseflies (42) to relatively high levels in cockroaches andfleas (9, 43). The high stability of these compounds may result inexposure to persistent residues in the environment, maintainingselection for the resistance allele in natural populations. Addi-tionally, increasing household and field use of phenylpyrazoleinsecticides, which also target the insect RDL GABA receptor, mayselect for resistance alleles (9, 44). In the early 1990s, theSer301 mutation frequency in populations of D. melanogaster inthe United States was estimated at 1% (45). This value is similarto the current estimate of 2.4% in the DGRP lines extractedfrom a population North Carolina and 3% in our survey ofAustralian populations (25) (SI Text).The duplication identified here creates adaptive potential for

accumulation of resistance mutations in one copy that may bedetrimental to the endogenous role of Rdl. Although the Ala301 toSer/Gly replacement does not result in lethality and in manyspecies has a negligible fitness effect (15), other replacements mayresult in enhanced resistance but have a greater impact on fitness.Ser301 provides low resistance to the phenylpyrazole fipronil thatdoes not impact the use of this insecticide in the field (9, 43, 46,47). However, fipronil-resistant strains isolated from two speciesof planthopper have been shown to contain an Ala to Asn, ratherthan Ser or Gly, mutation at the equivalent 301 site, found onlyin the heterozygous state (48, 49). The lack of homozygous Asnindividuals was proposed to be a result of lethality, supported bythe reduced GABA median effective concentration (EC50) ob-served in electrophysiological data (49). Mutations resulting inlethality can only be viable in a heterozygous state, or morepermanently, in a duplication of the type described in thisstudy. Continuing use of phenylpyrazole insecticides may resultin increased prevalence of Asn replacements and enhanced re-sistance in other relevant species. Duplications generating per-manent heterozygosity would facilitate the maintenance andspread of such mutations.

ConclusionDuplications are a major source of selectable genetic variationand provide evolutionary flexibility for adaptation to new func-tional niches. Here we characterize a recent duplication in a majorinsecticide target site, Rdl, in D. melanogaster. The duplicationis associated with insecticide resistance and RNA editing muta-tions in one copy and one WT copy. The resulting expression ofboth WT and mutant transcripts results in intermediate resistanceto dieldrin while reducing a temperature-sensitive fitness cost, cre-ating a functional, permanent, and heritable heterozygosity. Thisdiscovery highlights considerations for continued use of insecticidestargeting Rdl receptors in insects. A number of pest species exhibitRdl copy number variation and therefore have flexibility to accu-mulate resistance mutations while retaining sufficient WT func-tion. In species such as D. melanogaster, that are ancestrally singlecopy for Rdl, evidence of novel copy number variation at this targetsite provides a platform for future adaptation to environmentalpressures, such as the ongoing use of insecticides.

Materials and MethodsDrosophila melanogaster Lines. RdlR MD-RR (Bloomington: 1675) was origi-nally isolated in Maryland in 1990, with 4,000-fold dieldrin resistance, andwas used in the initial characterization of the Rdl Ser301 mutation (2, 6, 29).The sequence of Rdl from 168 DGRP genomes (25) was extracted using ref-erence genome coordinates, and polymorphisms were annotated manually.Sequence coverage and read depth of the Rdl genomic region was analyzedusing MAQ (http://maq.sorceforge.net/maq-man.shtml). Two DGRP lines wereused in phenotypic analysis of dieldrin resistance and heat shock recovery:one randomly selected RdlWT line, Ral-882, and one duplication line, Ral-378(Bloomington: 28255, 28187). An additional DGRP duplication line, Ral-318(Bloomington: 28168), was used alongside Ral-378 to genetically verify thepresence of a duplication by F1 analysis with w1118 (Bloomington: 3605),a control line containing WT Rdl, used in reciprocal crosses with the putativeduplication lines (Fig. S2). Transgenic integration of the Rdl locus was per-formed using the attB-P[acman]-ApR plasmid into chromosome III of theΦ-86Fb-attP line (Bloomington: 24749). The Φ-86Fb-empty line was used asa control for the two generated insertion lines: Φ-86Fb-P[ac]-RdlWT (WT Rdlinsertion) and Φ-86Fb-P[ac]-RdlA301S (Ser301 mutant Rdl insertion).

Generation of P[acman]-Rdl Lines. Recombineering was carried out accordingto the methods used in Venken et al. (50). A 55.7-kb fragment surroundingthe Rdl locus was obtained by the cloning of left and right arms (Table S3)into the attB-P[acman] vector and homologous recombination with BAC-1E12 (RP98 library, Drosophila Genomic Resources Centre). The Ala301 to Serreplacement was generated using the galK counter selection method (51).Both WT and A301S constructs were integrated into Φ-86Fb-attP, and trans-genics were identified by eye color and PCR confirmation. Expression of theSer301 transgene was detected by HaeII restriction digest of exon7 from cDNAgenerated from the Φ-86Fb-P[ac]-RdlA301S line.

Generation of RNA, cDNA, Cloning, and Sequencing. RNA was extracted from1-d-old adult heads from lines Ral-378 and Ral-318 using TRIzol reagent, andcDNA was synthesized with SuperScript III Reverse Transcriptase [oligo(dT)20;Invitrogen], following the manufacturer’s instructions. PCR primers weredesigned to amplify exon 7 and 8, spanning the two polymorphisms atcodons 301 and 360 (Rdl_Ex7_F/Ex8_R; Table S3). Products were cloned intopGEM T-easy (Promega) before sequencing (Macrogen).

Diagnostic HaeII Restriction Digest. The Ala301 to Ser polymorphism occursin exon 7 of Rdl as a result of a T to C base pair substitution, removing aHaeII restriction site (5); 268 bp of exon 7 was amplified (Rdl_Ex7_F/R (TableS3), and HaeII digest was used to differentiate between Ser301 (205 and63 bp), Ala301 (148, 57, and 63 bp), or heterozygous lines (205, 148, 63, and57 bp; Fig. S2).

Insecticide Screens. Dieldrin dosage–mortality analysis was carried out accord-ing to the same methods described in a DDT 24-h adult contact assay, wheredieldrin powder was dissolved in acetone and coated the inside of scintillationvials (52). Five replicates of at least five doses per strain were tested to generatedose–response curves using PriProbit (ver.1.63; Fig. 4) (53). Resistance ratios and95% CIs were estimated from dosage–mortality curves as previously described(54) (Table S6).

Fig. 6. Schematic showing a putative origin of the duplication in the Rallines, based on ectopic recombination of a heterozygous RdlR MD-RR/WTline, containing the nwk intron 7 Roo TE and the Glu-RIB intron 1 Roo TEin trans.

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Temperature Sensitivity Screens. Recovery from heat shock was conductedwith minor variations to tests performed in ref. 14. Flies were reared at roomtemperature (22–24 °C). Three replicates of 20 5- to 8-d-old adult femaleswere placed into glass vials equilibrated in a 38 °C water bath and heated for10 min. Unconscious flies were tipped onto a 2.5-cm-diameter circular arenaat room temperature, and flies departing from this area were scored every30 s for 15 min (Fig. 5). Proportional data were arcsine transformed, and

repeated-measures ANOVA was used to assess the significance of the effectsof genotype over time. A post hoc test using the Bonferroni correction wasused to compare differences between genotypes.

ACKNOWLEDGMENTS. We thank Tom Harrop for valuable comments on themanuscript and members of the Social Insects laboratory for advice on statistics.This research was funded by an Australian Research Council grant (to P.B.).

1. Georghiou GP (1986) The magnitude of the resistance problem. Pesticide Resistance:Strategies and Tactics for Management (National Academy Press, Washington, DC).

2. ffrench-Constant RH, Roush RT (1991) Gene mapping and cross-resistance in cyclodi-ene insecticide-resistant Drosophila melanogaster. Genet Res 57(1):17–21.

3. ffrench-Constant RH, Mortlock DP, Shaffer CD, MacIntyre RJ, Roush RT (1991)Molecular cloning and transformation of cyclodiene resistance in Drosophila:An invertebrate γ-aminobutyric acid subtype A receptor locus. Proc Natl Acad Sci USA88(16):7209–7213.

4. ffrench-Constant RH, Rocheleau T (1992) Drosophila cyclodiene resistance geneshows conserved genomic organisation with vertebrate γ-aminobutyric acidA receptors.J Neurochem 59(4):1562–1565.

5. ffrench-Constant RH, Steichen JC, Rocheleau TA, Aronstein K, Roush RT (1993) Asingle-amino acid substitution in a g-aminobutyric acid subtype A receptor locus isassociated with cyclodiene insecticide resistance in Drosophila populations. Proc NatlAcad Sci USA 90(5):1957–1962.

6. ffrench-Constant R, Rocheleau TA, Steichen JC, Chalmers AE (1993) A point mutationin a Drosophila GABA receptor confers insecticide resistance. Nature 363(6428):449–451.

7. Thompson M, Steichen JC, ffrench-Constant RH (1993) Conservation of cyclodieneinsecticide resistance-associated mutations in insects. Insect Mol Biol 2(3):149–154.

8. Andreev D, Kreitman M, Phillips TW, Beeman RW, ffrench-Constant RH (1999) Mul-tiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera:Tenebrionidae). J Mol Evol 48(5):615–624.

9. Bass C, Schroeder I, Turberg A, Field LM, Williamson MS (2004) Identification of theRdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis(Siphonaptera: Pulicidae). Pest Manag Sci 60(12):1157–1162.

10. Anthony N, Unruh T, Ganser D, ffrench-Constant R (1998) Duplication of the RdlGABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae. MolGen Genet 260(2–3):165–175.

11. Du W, et al. (2005) Independent mutations in the Rdl locus confer dieldrin resistanceto Anopheles gambiae and An. arabiensis. Insect Mol Biol 14(2):179–183.

12. ffrench-Constant RH, Pittendrigh B, Vaughan A, Anthony N (1998) Why are there sofew resistance-associated mutations in insecticide target genes? Phil Trans R Soc LondB 353(1376):1685–1693.

13. Zhang H-G, ffrench-Constant RH, Jackson MB (1994) A unique amino acid of theDrosophila GABA receptor with influence on drug sensitivity by two mechanisms.J Physiol 479(Pt 1):65–75.

14. ffrench-Constant RH, Steichen JC, Ode PJ (1993) Cyclodiene insecticide resistance inDrosophila melanogaster (Meigen) is associated with a temperature-sensitive phe-notype. Pesticide Biochem Physiol 46(1):73–77.

15. Aronstein K, Ode P, ffrench-Constant RH (1995) PCR based monitoring of specificDrosophila (Diptera: Drosophilidae) cyclodiene resistance alleles in the presence andabsence of selection. Bull Entomol Res 85(1):5–9.

16. Agosto J, et al. (2008) Modulation of GABAA receptor desensitization uncouples sleeponset and maintenance in Drosophila. Nat Neurosci 11(3):354–359.

17. Whitten MJ, Dearn JM, McKenzie JA (1980) Field studies on insecticide resistance inthe Australian sheep blowfly, Lucilia cuprina. Aust J Biol Sci 33(6):725–736.

18. McKenzie JA (1990) Selection at the dieldrin resistance locus in overwintering pop-ulations of Lucilia cuprina (Wiedemann). Aust J Zool 38(5):493–501.

19. Yu L-L, Cui YJ, Lang G-J, Zhang M-Y, Zhang C-X (2010) The ionotropic γ-aminobutyricacid receptor gene family of the silkworm, Bombyx mori. Genome 53(9):688–697.

20. Yuan G, Gao W, Yang Y, Wu Y (2010) Molecular cloning, genomic structure, andgenetic mapping of two Rdl-orthologous genes of GABA receptors in the diamondbackmoth, Plutella xylostella. Arch Insect Biochem Physiol 74(2):81–90.

21. Dale RP, et al. (2010) Identification of ion channel genes in the Acyrthosiphon pisumgenome. Insect Mol Biol 19(Suppl 2):141–153.

22. Bass C, Field LM (2011) Gene amplification and insecticide resistance. Pest Manag Sci67(8):886–890.

23. Raymond M, Chevillon C, Guillemaud T, Lenormand T, Pasteur N (1998) An overviewof the evolution of overproduced esterases in the mosquito Culex pipiens. PhilosTrans R Soc Lond B Biol Sci 353(1376):1707–1711.

24. Schmidt JM, et al. (2010) Copy number variation and transposable elements featurein recent, ongoing adaptation at the Cyp6g1 locus. PLoS Genet 6(6):e1000998.

25. Mackay TFC, et al. (2012) The Drosophila melanogaster genetic reference panel.Nature 482(7384):173–178.

26. Jones AK, et al. (2009) Splice-variant- and stage-specific RNA editing of the DrosophilaGABA receptor modulates agonist potency. J Neurosci 29(13):4287–4292.

27. Pool JE, et al. (2012) Population genomics of sub-saharan Drosophila melanogaster:African diversity and non-African admixture. PLoS Genet 8(12):e1003080.

28. Linheiro RS, Bergman CM (2012) Whole genome resequencing reveals natural targetsite preferences of transposable elements in Drosophila melanogaster. PLoS ONE 7(2):e30008.

29. ffrench-Constant RH, Roush RT, Mortlock D, Dively GP (1990) Isolation of dieldrinresistance from field populations of Drosophila melanogaster (Diptera: Drosophili-dae). J Econ Entomol 83(5):1733–1737.

30. Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesissystem for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad SciUSA 104(9):3312–3317.

31. Wilson TG (2001) Resistance of Drosophila to toxins. Annu Rev Entomol 46:545–571.32. Labbé P, et al. (2007) Independent duplications of the acetylcholinesterase gene

conferring insecticide resistance in the mosquito Culex pipiens. Mol Biol Evol 24(4):1056–1067.

33. Devonshire AL, Field LM (1991) Gene amplification and insecticide resistance. AnnuRev Entomol 36:1–23.

34. Wondji CS, et al. (2009) Two duplicated P450 genes are associated with pyrethroidresistance in Anopheles funestus, a major malaria vector. Genome Res 19(3):452–459.

35. Levasseur A, Pontarotti P (2011) The role of duplications in the evolution of genomeshighlights the need for evolutionary-based approaches in comparative genomics. BiolDirect 6:11.

36. Lynch M, Conery JS (2003) The origins of genome complexity. Science 302(5649):1401–1404.

37. Schmidt JM, Robin C (2011) An adaptive allelic series featuring complex generearrangements. PLoS Genet 7(10):e1002347.

38. Li X, Schuler MA, Berenbaum MR (2007) Molecular mechanisms of metabolic resistanceto synthetic and natural xenobiotics. Annu Rev Entomol 52:231–253.

39. González J, Petrov DA (2009) The adaptive role of transposable elements in theDrosophila genome. Gene 448(2):124–133.

40. de la Chaux N, Wagner A (2009) Evolutionary dynamics of the LTR retrotransposonsroo and rooA inferred from twelve complete Drosophila genomes. BMC Evol Biol9:205.

41. Montgomery EA, Huang S-M, Langley CH, Judd BH (1991) Chromosome rearrange-ment by ectopic recombination in Drosophila melanogaster: Genome structure andevolution. Genetics 129(4):1085–1098.

42. Gao J-R, et al. (2007) The A302S mutation in Rdl that confers resistance to cyclodienesand limited cross-resistance to fipronil is undetectable in field populations of houseflies from the USA. Pestic Biochem Physiol 88(1):66–70.

43. Kristensen M, Hansen KK, Jensen KM (2005) Cross-resistance between dieldrin andfipronil in German cockroach (Dictyoptera: Blattellidae). J Econ Entomol 98(4):1305–1310.

44. Cole LM, Nicholson RA, Casida JE (1993) Action of phenylpyrazole insecticides at theGABA-gated chloride channel. Pestic Biochem Physiol 46(1):47–54.

45. ffrench-Constant RH (1994) The molecular and population genetics of cyclodieneinsecticide resistance. Insect Biochem Molec Biol 24(4):335–345.

46. Brunet S, Le Meter C, Murray M, Soll M, Audonnet J-C (2009) Rdl gene polymorphismand sequence analysis and relation to in vivo fipronil susceptibility in strains of thecat flea. J Econ Entomol 102(1):366–372.

47. Ozoe Y, et al. (2000) Fipronil-related hetercyclic compounds: Structure-activity rela-tionships for interaction with γ-aminobutyric acid- and voltage-gated ion channelsand insecticidal action. Pestic Biochem Physiol 66(2):92–104.

48. Nakao T, Naoi A, Kawahara N, Hirase K (2010) Mutation of the GABA receptor as-sociated with fipronil resistance in the whitebacked planthopper, Sogatella furcifera.Pestic Biochem Physiol 97(3):262–266.

49. Nakao T, et al. (2011) The A2’N mutation of the RDL gamma-aminobutyric acid receptorconferring fipronil resistance in Laodelphax striatellus (Hemiptera: Delphacidae). J EconEntomol 104(2):646–652.

50. Venken KJT, He Y, Hoskins RA, Bellen HJ (2006) P[acman]: A BAC transgenic platformfor targeted insertion of large DNA fragments in D. melanogaster. Science 314(5806):1747–1751.

51. Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG (2005) Simple andhighly efficient BAC recombineering using galK selection. Nucleic Acids Res 33(4):e36.

52. Daborn P, Boundy S, Yen J, Pittendrigh B, ffrench-Constant R (2001) DDT resistance inDrosophila correlates with Cyp6g1 over-expression and confers cross-resistance to theneonicotinoid imidacloprid. Mol Genet Genomics 266(4):556–563.

53. Sakuma M (1998) Probit analysis of preference data. Appl Entomol Zool (Jpn) 33(3):339–347.

54. Robertson JL, Savin NE, Preisler HK, Russell RM (2007) Bioassays with Arthropods (CRCPress, Boca Raton, FL, 2nd Ed).

14710 | www.pnas.org/cgi/doi/10.1073/pnas.1311341110 Remnant et al.

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